Download Types of DNA Mutations - University of Minnesota

HIV Life Cycle
1 = Entry in CD4+ lymphocytes
2 = Reverse transcription
3 = Integration
4 = Transcription
5 = Translation
6 = Viral Assembly
Termination of Polymerization:
Nucleoside Drugs
NH2
O
NH
N
HN
N
O
HO
N
O
HO
N
N
N
N
O
NH2
N
HO
N
NH
N
NH2
O
HO
O OH
O
N3
OH
AZT
Ziagen
(zidovudine)
(abacavir)
Acyclovir
Antiviral
AraC
Antitumor
Other examples: dideoxycytidine, dideoxyinosine
Principle of action:
1) cellular uptake
2) activation to 5’-triphosphate
3) competition with normal substrate and
incorporation in DNA resulting in chain
termination
Nucleoside Drugs Must Be Converted to
Triphosphates to be Part of DNA and RNA
O
P O
HO
HO
HO
O
Base
Ki nase
ATP
O
OH
OH
Monophosphate
ATP
O
O
O
HO P O P O P O
HO
OH OH
O
OH
Base
Base
Ki nase
O
O
HO P O P O
HO
OH
ATP
Triphosphate
• Compete with normal substrate for RT binding
• Cause chain termination
Ki nase
O
OH
Diphosphate
Base
Termination of Polymerization:
Nucleoside Drugs
NH2
O
NH
N
HN
N
O
HO
N
O
HO
N
N
N
N
O
NH2
N
HO
N
NH
N
NH2
O
HO
O OH
O
N3
OH
AZT
Ziagen
(zidovudine)
(abacavir)
Acyclovir
Antiviral
AraC
Antitumor
Other examples: dideoxycytidine, dideoxyinosine
Principle of action:
1) cellular uptake
2) activation to 5’-triphosphate
3) competition with normal substrate and
incorporation in DNA resulting in chain
termination
DNA Chain termination by Nucleoside Analogs
Primer
Strand
O
O
P
Base
O
O-
O
Template
Strand
3'
OH
O
-O
P
O-
O
O
P
O-
O
O
P
O
Base
5'
O-
Mg 2+
Ziagen
No 3’OH!
Mechanisms of selectivity
1.
Activated drug is recognized and incorporated in
DNA only by reverse transcriptase, not by cellular
DNA polymerases (RNA viruses).
•
•
viral polymerases usually have lower fidelity
(no proofreading)
Mammalian DNA polymerases are more accurate
2. The drug is phosphorylated by viral kinase, not
by cellular kinases (e.g. AZT).
Mechanisms of resistance and possible solutions:
1.
2.
The drug cannot enter cells or is pumped out rapidly.
The drug is rapidly deaminated to inactive form or normal substrate is
overproduced.
3. The drug is no longer recognized by kinases and is not
activated to triphosphate form.
Possible solution:
Use activated phosphate form of nucleosides (Viread)
4. Activated drug is not incorporated in DNA by mutant reverse
transcriptase (usually HIV RT mutations at codons 184,65,69, 74, and 115).
Possible solution:
Use a mixture of several RT inhibitors (e.g. zidovudine (AZT) +
lamivudine (3TC) = Combivir®) or a mixture of different mechanisms
of action (e.g. non-nucleoside RT inhibitors, protease inhibitors).
Nucleoside inhibitors of DNA polymerase as
anticancer drugs
NH2
N
N
O
HO
O OH
OH
AraC (1--D-arabinofuranosylcytosine)
• used for treating acute myelocytic leukemia
• activated to triphosphate form by cellular kinases
• causes inhibition of DNA synthesis, repair, and DNA
fragmentation
• very toxic
DNA Synthesis in eukaryotes: Take Home
Message
1) DNA synthesis in eukaryotes is mechanistically similar to
replication in bacteria.
2) Eukaryotic replication is much slower and uses multiple
replication origins.
3) Chromosomal DNA is associated with histones which need to
be biosynthesized at the time of replication.
4) DNA Polymerases are more specialized, and their
interactions are more complex.
5) Chromosomal DNA is linear and thus requires special
processing of the ends.
6) Differences between mammalian and viral DNA polymerizes
can be exploited in treatment of viral infections.
DNA Damage, Mutations, and
Repair
See Stryer p. 768-773
DNA Mutations
1.
Substitution mutations: one base pair for another,
e.g. T for G
• the most common form of mutation
• transitions; purine to purine and pyrimidine to
pyrimidine
• transversions; purine to pyrimidine or pyrimidine to
purine
2. Frameshift mutations
•
Deletion of one or more base pairs
•
Insertion of one or more base pairs
Spontaneous mutations due to DNA
polymerase errors
•
•
Very low rate of misincorporation (1 per 108 - 1 per 1010)
Errors can occur due to the presence of minor tautomers
of nucleobases.
H3C
O
H2N
N
NH
N
N
N
N
O
T
amino
A
Rare imino tautomer of A
10-4
Normal base pairing
Mispairing
Consider misincorporation due to a rare tautomer of A
2nd
replication
1st
replication
5’ A
3’ T
A
T
A(imino)
C
A(imino)
T
G
C
A
T
Normal replication
Final result: A  G transition (same as T  C in the other strand)
Induced mutations result from DNA damage
Sources of DNA damage: endogenous
1. Deamination
2. Depurination: 2,000 - 10,000 lesions/cell/day
3. Oxidative stress: 10,000 lesions/cell/day
Sources of DNA damage: environmental
1. Alkylating agents
2. X-ray
3. Dietary carcinogens
4. UV light
5. Smoking
Normal base pairing in DNA and an example of
mispairing via chemically modified nucleobase
O
N
N
o
h
N
NH
h
N
NH2 O
G
OR
n
N
N
NH2
N
N
N
O
NH
HN
h
NH2
O
O6-AlkG
C
T
G A
G
C
G
T
A
T
DNA oxidation
Reactive oxygen species: HO•, H2O2, 1O2, LOO•
O
O
H3C
H3C
HO
NH
N
O
N
NH
N
N
O
thymine glycol
O
H
O
N
HO
NH
NH2
N
NH
O
N
N
NH2
8-oxo-G
•10,000 oxidative lesions/cell/day in humans
Deamination
NH2
N
O
N
N
N
N
N
A
O
N
N
G
N
NH2
O
N
N
Mechanism:
Hypoxanthine
N
HO
O
N
N
NH
N
NH2
NH
NH2
NH
N
H
Xanthine
N
O
N
N
H2O
N
- NH3
N
O
HO
NH2
N
NH
NH
O
N
C
O
N
N
N
Uracil
H NH
A
NH N
N
O
O
G
N
C
Rates increased by the presence of NO (nitric oxide)
N
NH
N
Depurination to abasic sites
O
N
O
O
O
N
O
NH
N
H2O
NH2
O
O
OH
O
Abasic site (AP
site)
2,000 – 10,000/cell/day
N
N
H
NH
N
NH2
UV light-induced DNA Damage
O
H3C
O
NH
NH
N
O
H3C
N
O
O
CH3
O
NH
O
O P OO
N
O
…CC…
O
O
O
CH3
NH
O
O P OO
N
O
O
Pyrimidine dimer
Easily bypassed by Pol  (eta) in an error-free manner
Deletions and insertions can be caused by intercalating agents
Stryer Fig. 27.44
Importance of DNA Repair
• DNA is the only biological macromolecule
that is repaired. All others are replaced.
• More than 100 genes are required for DNA repair,
even in organisms with very small genomes.
• Cancer is a consequence of inadequate DNA repair.
DNA Repair Types
• Direct repair
– Alkylguanine transferase
– Photolyase
• Excision repair
– Base excision repair
– Nucleotide excision repair
– Mismatch repair
• Recombination repair
Direct repair: O6-alkylguanine DNA alkyltransferase (AGT)

Directly repaires O6-alkylguanines (e.g. O6-Me-dG, O6-Bz-dG)
In a stoichiometric reaction, the O6 alkyl group is transferred to a
Cys residue in the active site. The protein is inactivated and
degraded.

O
N
N
CH3
O
N
N
N
NH
AGT-CH2-SH
NH2
O6-methylguanine
N
N
AGT-CH2-S
NH2
CH3
Excision Repair
Takes advantage of the double-stranded (double information)
nature of the DNA molecule.
Four major steps:
1. Recognize damage.
2. Remove damage by excising part of one DNA strand.
3. The resulting gap is filled using the intact strand as the template.
4. Ligate the nick.
Antiparallel DNA Strands contain the same
genetic information
5'
3'
5'
3'
5'
3'
3'
A :: T
A :: T
A :: T
G ::: C
G
G ::: C
T :: A
T :: A
T :: A
5'
Original DNA duplex
3'
5'
DNA duplex with
one of the nucleotides
removed
3'
5'
Repaired DNA duplex
Base excision repair (BER)
• Used for repair of small damaged bases in DNA (AP
sites, methylated bases, oxidized bases…)
H
N
O
N
N
O
O
O
OH
N
NH
NH
O
8-oxo-G
O
NH2
Abasic site (AP
site)
N
NH2
N
H
O
N
Xanthine
N
N
N
Me
N3-Me-Ade
• Human BER gene hogg1 is frequently deleted in lung
cancer
Nucleotide Excision Repair
•
Corrects any damage that both distorts the DNA molecule and
alters the chemistry of the DNA molecule (pyrimidine dimers,
benzo[a]pyrene-dG adducts, cisplatin-DNA cross-links).
O
H3C
O
NH
N
5'
O
O
O P OO
N
O
HO32N
CH
Pt
NH
H2N
N
Cl
Cl
O
O
3'
•
HO
O
NH
H2N N OHNH
O N
2
HOPt
OH H2N OH2
HO
OH
H
N
NH2
N
-GGH2N
H2N
N
Pt
N
O
N
N
NH
N
NH2
Xeroderma pigmentosum is a genetic disorder resulting
in defective NER
Mismatch Repair Enzymes
Nucleotide mismatches can be corrected after DNA synthesis!
Repair of nucleotide mismatches:
1. Recognize parental DNA strand (correct base) and daughter
strand (incorrect base)
Parental strand is methylated:
H3C
NH2
HN
N
N
N
O
N
Me
N
N
2. Replace a portion of the strand containing erroneous nucleotide
(between the mismatch and a nearby methylated site –up to 1000 nt)
Genetic diseases associated with defective DNA repair
Xeroderma Pigmentosum
NER
Hereditary nonpolyposis
colorectal cancer
MMR
Cockrayne’s syndrome
NER
Falconi’s anemia
DNA ligase
Bloom’s syndrome
BER, ligase
Lung cancer (?)
BER